The stability of individual proteins will be compared in exponentially growing and nongrowing cultures of Escherichia coli using pulse-chase experiments to follow the kinetics of protein degradation and modification. Presently the relative amounts of proteins separated by the two-dimensional gel electrophoresis method of O'Farrell are determined by a double-labeling procedure. In the near future, a computer-assisted method for analyzing autoradiograms will be tested for replacement of the double-labeling procedure. The identity of proteins separated by two-dimensional electrophoresis will be investigated in cooperation with Dr. Frederick C. Neidhardt. Proteolytic fragment analysis of individual proteins will be used to compare metabolically related proteins and to assist in unambiguously identifying proteins. The mathematical model developed in these studies to describe protein degradation and modification in exponentially growing and nongrowing cells will be extended to include multiple intermediate steps of modification. The model will be applied to results obtained in the kinetic pulse-chase experiments. The stability of individual proteins will also be examined in mutants defective in specific or general proteolytic systems. For example, mutants defective in protease I, II or III and mutants defective in abnormal protein degradation (deg mutants) will be studied. The stability of missense alpha subunits of tryptophan synthase that were unstable in growing or nongrowing cells according to enzymatic properties (Beta 2 subunit stimulation) will also be tested in normal strains and possibly in mutants defective in proteolytic systems. A cell-free system will be established to examine conditions necessary for degradation of specific proteins in vitro.